Phylogeography of Xiphorhynchus Fuscus (Passeriformes, Dendrocolaptidae): Vicariance and Recent Demographic Expansion in Southern Atlantic Forest

PHYLOGEOGRAPHY OF X. FUSCUS G. S. CABANNE ET AL .

Biological Journal of the Linnean Society, 2007, 91, 73–84. With 3 figures

Phylogeography of Xiphorhynchus fuscus (Passeriformes, Dendrocolaptidae): vicariance and recent demographic expansion in southern Atlantic forest

GUSTAVO SEBASTIÁN CABANNE1, FABRÍCIO R. SANTOS2 and CRISTINA YUMI MIYAKI1*

1Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, 05508-090, São Paulo, SP, Brazil 2Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, 31270-901, Belo Horizonte, MG, Brazil

Received 2 September 2005; accepted for publication 1 July 2006

Knowledge of the evolutionary processes that shaped a biota is important for both academic and conservation purposes. The objective of the present study is to analyse the mitochondrial genetic variation of Xiphorhynchus fuscus (Aves: Dendrocolaptidae) from the southern Atlantic forest in Brazil and Argentina, and to discuss whether the results support different hypotheses regarding the local intraspecific diversification of this species. We sequenced 575 bp of the control region of 114 specimens collected in the Brazilian states of Bahia, Minas Gerais, Rio de Janeiro, São Paulo, Paraná, and Santa Catarina, and in the province of Misiones in Argentina. We studied the population genetic structure with analysis of molecular variance and the demographic history with multiple regression analysis, coalescence simulations, and demographic tests. Xiphorhynchus fuscus presented a significant population genetic structure (Φst = 0.57). Three mitochondrial lineages were described, one associated with Xiphorhynchus fuscus tenuirostris and the others with Xiphorhynchus fuscus fuscus. The data did not support the primary influence of geographical barriers or rivers in the intraspecific diversification of X. fuscus in the southern Atlantic forest. Instead, the data supported the influence of isolation by geographical distance, recent vicariance events, and demographic expansions apparently related to Pleistocene and Holocene forest dynamics. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 73–84.

ADDITIONAL KEYWORDS: coalescence simulations – forest refuges – isolation by distance – mitochondrial control region – multiple regression – Neotropics – Pleistocene – riverine barriers – Valley of the Paraíba do Sul river.

The Atlantic forest is distributed along eastern Brazil, tas (Moritz et al., 2000), the evolution in palaeorefuges eastern Paraguay, and north-eastern Argentina and the inﬂuence of geographical barriers are two of (Gusmão Câmara, 2003). Its unique biota is probably the most discussed ones. the result of a complex evolutionary history; however, In the Neotropics, the refuge theory was originally few studies have attempted to clarify the biogeograph- proposed to explain speciation during the Pleistocene ical processes that shaped it (Mustrangi & Patton, mainly in the Amazon basin (Haffer, 1969; Vanzolini & 1997; Costa et al., 2000; Geise, Smith & Patton, 2001; Williams, 1970; Brown & Ab’Saber, 1979; Haffer & Pellegrino et al., 2005). The knowledge of these evolu- Prance, 2001). This theory proposes that during the tionary processes is important for both academic and glaciations the rainforests were reduced to refuges iso- conservation purposes (Moritz, 2002). Among the sev- lated by open areas, and that organisms isolated in eral hypotheses on the diversiﬁcation of rainforest bio- these refuges could have diverged and originated new lineages. Then, in the next interglacial period, the forest expanded and the new clades would be in contact. *Corresponding author. E-mail: [email protected] This hypothesis requires the expansion and contrac-

74 G. S. CABANNE ET AL. tion of open areas and the formation of forest refuges. river (VPSR). Those tectonic episodes could have been Palinological (Ledru et al., 1998; Behling & Negrelle, important for the biota in the southern Atlantic forest, 2001; Behling, 2002) and other types of studies (Brown where a complex relief exists with many mountain & Ab’Saber, 1979) propose that open areas dominated ranges and valleys, and where neotectonic activity the Atlantic forest’s landscape during the maximum was described (Petri & Fulfaro, 1983; Riccomini et al., of Late Pleistocene glaciations, suggesting that the 1989). refuge theory can be important to understand the Xiphorhynchus fuscus is an Atlantic forest endemic biological diversification of the biome. member of the Family Dendrocolaptidae (Aves) with a Pellegrino et al. (2005), based on a phylogeographi- broad distribution. It occurs in eastern Brazil (from cal study of the gecko Gymnodactylus darwinii the state of Ceará to the state of Rio Grande do Sul), (Gekkonidae, Squamata), proposed that rivers play an eastern Paraguay and north-eastern Argentina important role in the diversification of Atlantic forest (Marantz et al., 2003). There are four subspecies biota. Other authors proposed that the tectonic activ- defined by slight variations in colour and morpholo- ity associated with the formation of geographical land- gical measurements (Marantz et al., 2003). Xipho- marks could be relevant to biodiversity modelling, as rhynchus fuscus tenuirostris and Xiphorhynchus suggested by Silva & Straube (1996) who observed fuscus fuscus inhabit the eastern and the southern that the geographical range of some passerines was range of the species, respectively (Fig. 1). The other limited by a graben, the valley of the Paraíba do Sul subspecies inhabit the interior of the Brazilian state of

Figure 1. Study area, sampling localities, and distribution of Xiphorhynchus fuscus subspecies and mtDNA lineages. Locality numbers correspond to Table 1. BA, Bahia; ES, Espírito Santo; GO, Goiás; MG, Minas Gerais; MI, Misiones; MT, Mato Grosso do Sul; PR, Paraná; RJ, Rio de Janeiro; RS, Rio Grande do Sul; SC, Santa Catarina; SP, São Paulo. Approximate distribution limits of the two subspecies indicated by broken lines (Zimmer, 1947; Marantz et al., 2003). A: X. fuscus tenuirostris, B: X. fuscus fuscus.

PHYLOGEOGRAPHY OF X. FUSCUS 75

Table 1. Localities, sample sizes (N), haplotypes, and nucleotide diversity (π) in percentage

Locality N Haplotypesj π% (SD%)

1. Minas Gerais (MG), Jequitinhonha, left bank of the 5a T21, T41, T61, T72 0.5186 (0.2198) Jequitinhonha river, 16°20′S, 41°00′W 2. MG, Salto da Divisa, left bank of the Jequitinhonha river, 6a T14, T51, T71 0.4606 (0.1785) 16°05′S, 40°02′W 3. Bahia, Porto Seguro, 17°22′S, 40°17′W1b T31 – 4. MG, Nova Lima, 19°59′S, 43°49′W1a N81 – 5. MG, Marliéira, 19°43′S, 42°44′W1a N91 – 6. MG, Caratinga, 20°50′S, 42°05′W1a N121 – 7. MG, Simonésia, 20°07′S, 42°00′W1a N131 – 8. MG, Araponga, 20°40′S, 42°31′W3a N21, N101, N111 0.4710 (0.2374) 9. Rio de Janeiro, Itatiaia National Park, 22°25′S, 44°36′W10N26, N61, N141, N152 0.2274 (0.1239) 10. São Paulo (SP), Bananal State Park, 22°41′S, 44°19′W2c N31, N61 0.7109 (0.3368) 11. SP, Picinguaba, 22°31′S, 44°50′W4N24 0.0 12. SP, Caraguatatuba, 23°37′S, 42°26′W2N21, N141 0.1760 (0.1771) 13. SP, Morro do Diabo State Park, 22°30′S, 52°18′W1S11 – 14. Paraná (PR), Ortigueira, 24°12′S, 50°55′W1S81 – 15. PR, Wenceslau Braz, 22°51′S, 49°47′W7d S15, S82 0.1677 (0.1237) 16. SP, Barreiro Rico, 22°38′S, 48°13′W3e N22, S81 0.9479 (0.3195) 17. SP, Itaberá State Park, 23°51′S, 49°08′W7S43, S63, S101 0.2014 (0.1154) 18. SP, Burí State Park, 23°39′S, 48°32′W9f S44, S71, S83, S91 0.1760 (0.1098) 19. SP, Caboclos, 24°28′S, 48°35′W8S45, S83 0.0943 (0.0949) 20. SP, São Roque, 23°34′S, 47°09′W9N13, N21, S45 0.9199 (0.2815) 21. SP, Morro Grande State Park, 23°42′S, 46°59′W14N15, N23, S51, S62, S73 0.8830 (0.2589) 22. SP, Juquitiba, 23°53′S, 47°00′W7g N11, N42, S41, S61, S71, S81 1.0172 (0.3025) 23. SP, Serra do Mar State Park, Station Curucutú, 23°58′S, 4h N21, N41, N51, N71 0.4424 (0.1858) 46°44′W 24. Argentina, Misiones (MI), Campo San Juan State Park, 27°22′S, 3S11, S22 0.1173 (0.1065) 55°39′W 25. MI, Yabotí Biosphere Reserve, 26°48′S, 53°55′W3S12, S31 0.1173 (0.1181) 26. Santa Catarina, Botuverá, 27°13′S, 49°03′W1i S11 – aTissue samples deposited at the Universidade Federal de Minas Gerais; all the other samples are deposited at the Universidade de São Paulo. Samples with museum voucher or specimen ﬁeld (f) numbers: bMuseu Paraense Emílio Goeldi fAA568; cMuseu de Zoologia da Universidade de São Paulo (MZUSP) f76, f91; dMZUSP fITA257, fITA302, f ITA283; eMZUSP f31, f59, f60, fMUZUSP 76134, MZUSP 75588; gMZUSP fITA141, fITA171, fITA182, fITA152, fITA172, fITA157, 75565; hMZUSP f9; iMZUSP 75032. jNumbers in superscript indicate the number of individuals with the corresponding haplotype.

Bahia (Xiphorhynchus fuscus brevirostris) and the MATERIAL AND METHODS north-eastern part of the country (Xiphorhynchus fuscus atlanticus). The distribution limits of these sub- STUDY AREA AND SAMPLES species are not well known. Given its geographical Samples (blood or muscle, N = 114) were collected distribution and deep-forest dependency, X. fuscus is a between 2000 and 2004 in the Brazilian states of good model to study the diversification of the Atlantic Bahia, Minas Gerais, Rio de Janeiro, São Paulo, forest biota. Paraná, and Santa Catarina, and in the province of The objective of the present study is to analyse Misiones in Argentina (Fig. 1, Table 1). The collection the mitochondrial DNA variation of populations of localities are covered by dense ombrophilus, mixed or X. fuscus from the southern part of the Atlantic forest semideciduous forest, which are the main forest types and to discuss whether the results support different of the Atlantic forest (Veloso, 1991). The relief in the hypotheses regarding the local intraspecific genetic study area is complex, especially in the eastern por- diversification of this bird. tion due to the presence of the Serra do Mar and the

Serra da Mantiqueira coastal ridges, which can sur- used in the phylogeographical analyses. Identification pass 2000 m a.s.l. and simulation of the secondary structure of the tRNA Blood was collected (approximately 0.1 mL) with sequences were carried out with the program insulin syringes from the largest vein in the right cer- tRNAscan-SE 1.21 (http://www.genetics.wustl.edu/ vical region. Muscle was obtained from specimens eddy/tRNAscan-SE/Lowe & Eddy, 1997). All sequences which were deposited at the Museu de Zoologia da are deposited in GenBank under the access numbers Universidade de São Paulo. Each bird was captured AY948386–AY948394, AY948396–AY948415, and with mist nets, photographed and marked with an alu- DQ144622–DQ144636. minium ring. All tissue samples are deposited at the Sequences were aligned with the program Clustal X Laboratório de Genética e Evolução Molecular de Aves (Thompson et al., 1997). The likelihood ratio test as (Instituto de Biociências, Universidade de São Paulo, implemented in the software Modeltest, version 3.7 Brazil), or at the Laboratório de Biodiversidade e (Posada & Crandall, 1998), was used to select the best Evolução Molecular (Instituto de Ciências Biomédicas, fit evolutionary model [HKY85 with the proportion of Universidade Federal de Minas Gerais, Brazil). invariable sites (I) of 0.8404 and a discrete gamma distribution (α = 0.6467); Hasegawa et al., 1985]. We MOLECULAR METHODS reconstructed the phylogenies by Neighbour-joining (NJ; Saitou & Nei, 1987) with the software PAUP*, Total DNA was obtained from blood or muscle samples version 4.0b10 (Swofford, 2001) and by maximum like- by a conventional proteinase K–SDS digestion, lihood (ML) with PHYML Online (Guindon & Gascuel, organic extraction with phenol–chloroform, and etha- 2003; Guindon et al., 2005). The starting tree for the nol precipitation (Bruford et al., 1992). To diminish ML analysis was obtained with PHYML Online. For the possibility of amplifying unexpected copies of both analyses, the best fit evolutionary model was nuclear mtDNA inserts, we amplified an appro- used. A sequence of Xiphorhynchus pardalotus (tissue ximately 1600-bp fragment with the primer pair IBUSP P264) was used to root the resulting trees. The LXfB2 (5′-TCAATTCCAAACAAACTAGGAGG-3′, pre- support of the nodes was evaluated by nonparametric sent study)/HPRO (5′-GCTTTGGGAGTTGGAGATAA bootstrap (100 replicates; Felsenstein, 1985). AGG-3′, present study). This amplicon contained the In all further population analyses, we used the complete control region (total size of 1275 bp, deter- Tamura & Nei (1993) model of evolution, which is the mined in the present study). The PCR reaction (10 µl) closest one to the HKY85 model available in the soft- contained 20–40 ng of total DNA, 1X of Taq buffer ware we used. The haplotype network was constructed (Pharmacia Biotech), 200 µM of each dNTP, 1 µM of in accordance with Templeton, Crandall & Sing (1992) each primer, and 0.1 U of Taq polimerase (Pharmacia with the program TCS, version 1.13 (Clement, Posada Biotech). Amplifications were performed with an ini- & Crandall, 2000). The net divergence between sets tial step at 95 °C for 4 min and 37 cycles of 45 s at of sequences was obtained in MEGA, version 3.0 94 °C, 45 s at 53.5 °C, and 110 s at 72 °C, followed by a (Kumar, Tamura & Nei, 2004) with the formula final extension of 10 min at 72 °C. PCR products were δ = δ − [0.5(δ + δ )], where δ is the mean divergence purified with shrimp alkaline phosphatase and exonu- xy x y xy between all the sequences, and δ and δ are the mean clease I. Sequencing reactions were performed with x y divergences within each group of sequences x and y, Big Dye Terminator Kit, version 3.0 (Applied Biosys- respectively (Wilson et al., 1985). The mutation rate tems Inc.) using the amplification and the internal used to estimate the divergence time between the lin- primers (HXfRC1 5′-GGGGAAAATAAACGTTTATTA eages was µ = 8.72 × 10−8 changes per nucleotide, as AGTG-3′, HXfRC2 5′-CAAGATGGACATGTTCGAC used by Milot, Gibbs & Hobson (2000) for the analysis ACCG-3′, present study, and L537 5′-CCTCTGGTTC of the first domain of the mtDNA control region of the CTCGGTCAG-3′; Sorenson et al., 1999). The sequenc- passerine Dendroica petechia. We assumed a genera- ing reactions were precipitated with isopropanol 75% tion time of 1 year (Klicka & Zink, 1999). and etanol 70%, and analysed in an automatic To test whether the X. f. fuscus gene genealogy is sequencer ABI Prism 377 (Applied Biosystems Inc.). the result of the neutral coalescence process within a panmictic population, we followed Knowles (2001) and ANALYTICAL METHODS applied a gene-tree population-tree approach using The complete control region (1275 bp) and flanking the program MESQUITE 1.02 (Maddison & Maddi- genes (tRNAThr 70 bp, tRNAPro 70 bp) from one son, 2004). In this approach, gene trees (300 repli- X. f. fuscus sample (tissue IBUSP P652) were cates) were simulated by neutral coalescence under a sequenced. The analysis of 1048 bp of the control null model of a unique panmictic X. f. fuscus popula- region (positions 57–1104) from 20 individuals tion whose effective population size (Ne) was invari- revealed that the most variable portion encompasses able until total coalescence. Four arbitrary Ne were 575 bp (positions 57–631); thus, this segment was tested: 100 000, 50 000, 25 000, 12 000, and 6000. The

© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 73–84 PHYLOGEOGRAPHY OF X. FUSCUS 77 discordance between these gene trees and the alterna- Demographic expansion was inferred by calculating tive model of two isolated populations (one in the Fs (Fu, 1997) and R2 (Ramos-Onsins & Rozas, 2002) north and the other in the south of the Valley of the using DnaSP, version 4 (Rozas & Sánchez-Del Barrio,

Paraíba do Sul river, VPSR; Fig. 1) was measured 2003). Significantly negative Fs and low values of R2 using statistic s (Slatkin & Maddison, 1989). The dis- indicate demographic expansion. For these calcula- cordance between the reconstructed gene tree (NJ tree tions, the three major lineages recovered in the gene- of X. f. fuscus sequences) and the two populations’ alogy were defined as populations, following Cheviron, model was also evaluated with the statistic s and this Hackett & Caparella (2005). Significance was deter- value was compared with the expected distribution of mined based on 1000 coalescent simulations under a s-values obtained from the neutral simulations. If the model of population stability using empirical sample s-value of the reconstructed gene tree is significantly sizes and estimates of Θ. The parameters of exponen- lower than the values from the simulated gene trees tial population growth (Θ0, Θ1, and τ), according to the (α = 0.05), the null model is rejected. We used the model of Rogers & Harpending (1992), were estimated nucleotide diversity (π, Nei & Kumar, 2000) calculated in the program Arlequin, version 2.0. The expansion in Arlequin, version 2.0 (Schneider, Roessli & time was estimated according to t = τ/2u using lower Excoffier, 2000) to approximate theta (Θ) and to and upper values of 95% confidence interval of the calculate the empirical Ne according to the relation- parameter τ, where u is the mutation rate per gene- ship π = Θ = 2Neµ (Watterson, 1975; Nei & Kumar, ration per haplotype (Rogers & Harpending, 1992). 2000). We estimated the nucleotide diversity for each local- RESULTS ity where two or more individuals were available using Arlequin 2.0. The analysis of molecular variance CHARACTERISTICS OF THE SEQUENCES (AMOVA; Excoffier, Smouse & Quattro, 1992) was The sequences obtained revealed evidence of being of used to study the population genetic structure as mitochondrial origin: (1) the empirical base composi- implemented in Arlequin, version 2.0. We performed tion of the sequences used in the phylogeographical global (all the localities) and regional analyses (sub- study (A, 27.7%; C, 25%; G, 16.3%; and T, 31%) is com- Φ groups of localities). The ST was obtained using cor- patible with the expected one for avian mitochondrial rected distances and its statistical significance was DNA (Baker & Marshall, 1997); (2) the amplicon estimated by a nonparametric permutation test (1600 bp) contained the complete control region (Excoffier et al., 1992). (1275 bp), flanked by a tRNAThr at the 5′ end (70 bp) We performed a partial regression analysis and a tRNAPro at the 3′ end (70 bp), and this gene (Smouse, Long & Sokal, 1986) to test the effect of two arrangement is the expected one for Suboscines independent variables (linear geographical distance (Mindell, Sorenson & Dimcheff, 1998); (3) the inferred among the pairs of localities and their geographical secondary structure and the presence of expected anti- location) on the average corrected genetic distances codons (UGU and UGG) suggested that these tRNA among individuals from pairs of locations. Genetic dis- genes are functional (data not shown); and (4) the tances were estimated with the program MEGA, ver- amplicon analysed is longer than the average size of sion 3.0. The matrix of geographical location was translocated copies of mitochondrial genes in an avian constructed as a dummy variable (Quinn & Keough, nuclear genome (Pereira & Baker, 2004). The 575 bp of 2002) that indicated whether each pair of localities the control region of 114 individuals of X. fuscus pre- were within the same main geographical region sented 32 haplotypes (Table 1, Figs 2, 3), with 31 poly- (north-western Minas Gerais plus southern Bahia, morphic sites (5.39% of all positions), 30 transitions south-eastern Minas Gerais plus southern Rio de Jan- and two transversions, and no indels. eiro, and south from the VPSR). The magnitude of the partial regression coefficients indicates the relative importance of the different independent variables. If GENEALOGY, DIVERGENCE, AND GEOGRAPHICAL genetic distances reflect simple isolation by distance, DISTRIBUTION OF X. FUSCUS MITOCHONDRIAL geographical distance would be the best predictor. If LINEAGES vicariance were the most important process, the geo- The haplotype network presented three major clades graphical location matrix would be the best predictor (Fig. 2): one associated with X. f. tenuirostris and the of genetic distances. Each matrix was transformed to a others with X. f. fuscus [northern lineage (N) and mean of zero and a variance of one before the analyses southern lineage (S)]. The X. f. tenuirostris lineage (Smouse et al., 1986). The partial regression analyses occurred in samples from north-eastern Minas Gerais were performed in the program Fstat, version 2.9.3.2 and southern Bahia. Haplotypes of the X. f. fuscus (Goudet, 2002), using 10 000 permutations to obtain northern lineage only occurred in south-eastern Minas the probability values. Gerais, southern Rio de Janeiro and north-eastern São

S4S4

S5S5 S7S7 S1S1 S8S8 S6S6

S2S2 S3S3 S9S9 S10S10

N7N7

N6N6

N8N8

N14N1N14 N15N1N15

N12N1N12 N2N2 T5T5 T7T7 N10N1N10 N3N3 N5N5 N13N1N13 T3T3 N9N9 N11N1N11 N4N4 T1T1 T2T2 N1N1 T4T4 T6T6

Figure 2. Haplotype network of Xiphorhynchus fuscus based on 575 bp of the control region. The size of each ﬁgure is proportional to the frequency of the corresponding haplotype in the total sample. Haplotype identiﬁcation: T1 to T7, Xiphorhynchus fuscus tenuirostris lineage; N1 to N15, X. f. fuscus northern lineage; S1 to S10, X. f. fuscus southern lineage.

Paulo; whereas sequences of the southern lineage (135 000 ± 32 000 years) was based on the mean dis- occurred in all localities in southern and central São tance between groups (2.368 ± 0.561%). Paulo, Paraná, Santa Catarina, and Misiones. Based on coalescence principles, the TCS algorithm (Cran- dall & Templeton, 1996) selected the haplotype S4 COALESCENCE SIMULATIONS (X. f. fuscus lineage S) as the most likely ancestral one. Sequences within X. f. fuscus grouped in two main The phylogenetic analysis by NJ and ML resulted in lineages that were strongly associated with speciﬁc similar topologies and recovered the same three main geographical regions, suggesting that each lineage lineages. Thus, we only present the NJ tree with the possibly evolved in allopatry. Given that the diver- bootstrap supports obtained by both methods (Fig. 3). gence between lineages is low and that the stochastic Mean ± SD Tamura & Nei (1993) distance between nature of the coalescence process can produce many X. pardalotus and X. fuscus was 12.366 ± 1.481%. The different genealogies, we applied a gene-tree popula- distance between X. f. fuscus and X. f. tenuirostris was tion-tree analysis to test whether a single historical 2.368 ± 0.561%, and between X. fuscus lineages N population could have originated these results. The and S was 1.493 ± 0.400%. The net divergence s-value (nine) of the reconstructed genealogy was between the two X. f. fuscus lineages was 1.228 ± signiﬁcantly lower than the values obtained by the

0.432%, which corresponds to 73 500 ± 24 500 years of simulated gene trees, regardless of the Ne used divergence. The low number of samples of X. f. (P < 0.01); thus, the null model of a single population tenuirostris did not allow us to estimate the net diver- was rejected. Also, the empirical Ne was 52 000 and is gence between this clade and X. f. fuscus; thus, the compatible with the range of Ne (6000−100 000) estimated divergence time between these clades used in the simulations.

POPULATION GENETIC STRUCTURE

A global AMOVA indicated that 57.7% (ΦST = 0.577, P < 0.01) of the genetic variation in the samples of X. fuscus is allocated among the geographical regions where the main lineages occur (north-eastern Minas Gerais plus southern Bahia, south-eastern Minas Gerais plus southern Rio de Janeiro, and south of the VPSR). A regional AMOVA without the samples from the X. f. tenuirostris lineage (north-eastern Minas Gerais and southern Bahia) was performed subdivid- ing each main region in two subregions. The subregions of the ﬁrst region are Minas Gerais and Rio de Janeiro, whereas the subregions of the second region are São Paulo and south of São Paulo. This analysis indicated that 30.84% (P < 0.01) of the genetic diversity within X. f. fuscus was found among regions, that 12.93% (P < 0.01) occurred among the subregions within regions, and that 56.23% (P < 0.01) was allo-

cated within subregions. The ΦST of this analysis was 0.43.

RELATIONSHIPS BETWEEN GENETIC DISTANCES AND PREDICTOR VARIABLES The partial regression analysis indicated that 40.5% of the variation in genetic distances can be predicted by the linear geographical distance among pairs of localities (partial regression coefﬁcient = 0.585, P < 0.01) and the geographical location (partial regression coefﬁcient = 0.251, P < 0.01).

HISTORICAL DEMOGRAPHY

The Fs value for X. f. tenuirostris is nonsigniﬁcant, suggesting that there was demographic stability

(Table 2). The Fs values are signiﬁcant and negative for both X. f. fuscus lineages, suggesting the presence

of past demographic expansion. Because the R2 test is more suitable for the analysis of small samples than

the Fs test (Ramos-Onsins & Rozas, 2002), we analy-

sed X. f. tenuirostris (N = 12) using the R2 test. The result was nonsignificant (R2 = 0.1800, P = 0.6960), indicating that this taxa presented past demographic τ Figure 3. Neighbour-joining (NJ) tree based on 575 bp of stability. Based on the estimations of , the demo- the control region of Xiphorhynchus fuscus. The numbers graphic expansions of the northern and southern lin- at nodes show NJ and maximum likelihood bootstrap val- eages of X. f. fuscus started approximately 57 000 and ues above 50%, respectively. Xiphorhynchus pardalotus 19 000 years ago, respectively. was used to root the tree. Haplotype identification: T1 to T7, Xiphorhynchus fuscus tenuirostris lineage; N1 to N15, DISCUSSION X. f. fuscus northern lineage; S1 to S10, X. f. fuscus southern lineage. PHYLOGEOGRAPHICAL STRUCTURE OF X. FUSCUS IN THE SOUTH-EASTERN ATLANTIC FOREST The study of the control region of X. fuscus revealed a significant population genetic structure, which is in accordance with other Neotropical birds (Bates, 2000,

Table 2. Historical demographic analysis and expansion dates in years ago

Clade NFs* P (Fs)* Θ0 Θ1 Expansion dates

Xiphorhynchus fuscus tenuirostris 12 −1.400 0.160 NA NA NA Xiphorhynchus fuscus fuscus N46−7.080 0.001 0.0010 7.0460 10 130–57 370 Xiphorhynchus fuscus fuscus S56−3.789 0.015 0.0000 3415 4 690−19 460

N, sample size; P (Fs), probabilities of the Fs statistic value being lower than the observed one based on 1000 coalescent simulations; Θ0 and Θ1, parameters of the model of exponential growth of Rogers & Harpending (1992); NA, not applicable.

*Fs was calculated sensu (Fu, 1997).

2002; Aleixo, 2004; Cheviron et al., 2005), and con- Jequitinhonha rivers, is compatible with patterns trasts with the patterns observed with the same observed in other organisms. For example, small genetic marker in Paleartic passerine populations sep- mammals present biogeographical divergence among arated by thousands of kilometres (Merilä, Björklund north-eastern Minas Gerais, south-eastern Minas & Baker, 1997; Kvist et al., 1998; Kvist et al., 1999a, b; Gerais, and southwards regions (Mustrangi & Patton, Uimaniemi et al., 2003). 1997; Costa et al., 2000). In the gecko G. darwinii Three main mitochondrial lineages were revealed in (sampled in the same geographical region studied the X. fuscus populations studied. One lineage was here), Pellegrino et al. (2005) also found three main associated with the subspecies X. f. tenuirostris and mitochondrial lineages. Geckos from southern Minas the others with the subspecies X. f. fuscus (Figs 1, 2, 3). Gerais are divergent from those from São Paulo and Haplotypes of X. f. fuscus northern lineage were found southwards regions, and both clades are also sepa- from the Doce river basin in Minas Gerais to north- rated from geckos from the Jequitinhonha river basin. eastern São Paulo. The other X. f. fuscus lineage was Thus, X. fuscus and G. darwinii present similar phy- distributed from northern São Paulo to Santa Catarina logeographical patterns. Even considering that coales- and Misiones in Argentina. The two subspecies clades cence times present high variance and estimated appear to have diverged in the late Pleistocene, around divergence times may not be reliably compared, the 130 000 years ago. The separation of the two difference between the splitting times among clades in X. f. fuscus clades was estimated to have occurred the gecko (youngest divergence at least 0.9 Mya) and approximately 70 000 years ago. A contact zone those among the bird clades (oldest divergence between the two clades of X. f. fuscus was located to the 130 000 years ago) does not support a common tempo- south-west of VPSR (Fig. 1). We hypothesize that a ral origin of these patterns. Furthermore, Pellegrino contact zone between the X. f. tenuirostris clade and et al. (2005) suggested that rivers were the barriers the northern X. f. fuscus lineage can occur along the generating the phylogeographical pattern observed in Doce river in Espírito Santo and somewhere between the gecko. Some predictions for the hypothesis of riv- the basins of the Jequitinhonha and the Doce rivers in ers as primary gene flow barriers are that sister clades the interior of Minas Gerais, as suggested by the dis- should occur across major rivers rather than along the tribution of these two subspecies (Fig. 1). Further same river bank and should not present signals of studies are needed to test whether the Doce river can demographic expansion, which would favour second- be a barrier to gene flow between the two subspecies. ary contact (Moritz et al., 2000). The samples that we Another interesting result is that all southern locali- had available did not permit these predictions to be ties (Misiones and Santa Catarina) present only three tested in all the main river systems of the study area. haplotypes (S1, S2, and S3). These haplotypes are For the area around the Paraíba do Sul river, the num- grouped in the network and in the NJ tree and were ber of samples allowed us to start to explore this issue. only present in this region and in northern Paraná Because the two X. f. fuscus lineages presented signals (Table 1). This suggests that there was a past range of demographic expansion (Table 2) and the northern fragmentation somewhere between São Paulo and lineage occurs on both sides and southwards of the northern Paraná, and this needs to be tested in future VPSR (Fig. 1), this river does not appear to be a pri- studies with a more detailed sampling. mary barrier for the gene flow of X. fuscus. Neverthe- Even though studies on Atlantic forest organisms less, the limits of the distribution of the subspecies are scant, the biogeographical pattern observed in X. f. tenuirostris and X. f. fuscus appear to coincide X. fuscus with three phylogeographical groups that with the Doce river (Fig. 1), especially in Espírito appear to be limited by geographical landmarks or Santo, suggesting that this river could be a barrier. It specific geographical regions, such as the VPSR and is necessary to add more samples from key locations to the transition between the basins of the Doce and the test these hypotheses.

ON THE ORIGIN OF THE PHYLOGEOGRAHIC STRUCTURE formation of the valley. Thus, our data do not support OF X. FUSCUS the formation of the current VPSR as the vicariant event that produced the two X. f. fuscus lineages. The partial regression analysis suggested that both Brown & Ab’Saber (1979) suggested that the forest isolation by geographical distance and the history of cover along the VPSR was not discontinued in the last vicariance (geographical location) were important in glacial period. Based on this hypothesis, Silva & shaping the population genetic structure of X. fuscus. Straube (1996) suggested that the putative forest frag- The coalescence simulations supported the idea that mentation that resulted in the distribution pattern of X. f. fuscus lineages evolved in two populations L. falcinellus and L. squamatus could not be related to instead of in a single one. At least two main vicariant this historical climatic oscillation. Clapperton (1993), phenomena were detected: one that separated the two however, suggested that, in the last glacial maximum, subspecies lineages and the other which resulted in there were two main forest refuges in this area: one in the divergence between the two X. f. fuscus phylo- the northern Serra do Mar and the other in the Serra groups. The latter divergence is focussed on below. da Mantiqueira and part of the Serra do Espinhaço in Evolution in isolation and secondary contact pro- Minas Gerais; and these refuges were separated by vides a possible explanation for the phylogeographical a grassland area coincident with the VPSR. Under structure of X. f. fuscus. Two vicariant events could the scenario of Clapperton (1993), the palaeorefuge have separated the ancestral population: the geologi- hypothesis could explain the diversification of clades cal episodes that formed the VPSR or natural forest in X. f. fuscus, and thus the current contact area of the fragmentation. X. f. fuscus lineages should be secondary (Moritz et al., The VPSR is a 173 km long and up to 2 km deep gra- 2000). A prediction of this hypothesis is that clades ben that separates the Serra da Mantiqueira and the involved in the secondary contact should exhibit evi- northern Serra do Mar (Petri & Fulfaro, 1983) (Fig. 1). dence of range expansion (Hewit, 2000; Moritz et al., It is a contact area for birds (i.e. Lepidocolaptes 2000; Cheviron et al., 2005) and concomitant demo- squamatus and Lepidocolaptes falcinellus, Silva & graphic expansion. The demographic analysis sup- Straube, 1996; Heliobletus contaminatus contamina- ports this prediction for the two X. f. fuscus lineages. tus and Heliobletus contaminatus camargoi; Silva & Furthermore, the contact area between X. f. fuscus lin- Stotz, 1992) and marsupials (i.e. Marmosops paulensis eages has a high proportion of derived haplotypes, and Marmosops incanus; Mustrangi & Patton, 1997), especially from the northern lineage (i.e. haplotypes and it separates mitochondrial lineages of the gecko N1, N4, and N5; Table 1, Fig. 2), which is also compat- G. darwinii (Pellegrino et al., 2005). Also, morpholog- ible with population expansion and secondary contact ically distinct populations of the passerine Scytalopus (Templeton, Routman & Phillips, 1995). speluncae (Giovanni, 2005) come into contact in the According to Behling (1998, 2002), grassland domi- same geographical sector where the two X. f. fuscus nated the southern and south-eastern Brazilian land- lineages meet. Silva & Straube (1996) postulated that scape during the Late Pleistocene, where diverse forest the tectonic process that opened the valley and the ecosystems exist today. The current southern limit of consequent alteration of the forest cover could have the Atlantic forest biome contacts the grasslands biome been a significant vicariant event for forest species. of southern Brazil at latitude 28–27°S in the north of Assuming that this proposition is correct, the two lin- Rio Grande do Sul, and Behling (2002) concluded that, eages of X. f. fuscus that meet near to the valley could during the Late Pleistocene, this limit extended 750 km have evolved in isolation after the formation of the val- northwards, at least to latitude 20°S (southern Minas ley and, when the conditions for dispersion improved, Gerais and northern São Paulo). Behling (2002) also they came into contact. The predictions of this hypoth- proposed that the modern forest cover of south and esis are that populations at each side of the valley are south-east Brazil was established only in the Late differentiated, and that the separation time between Holocene. Under this historic scenario of dynamic forest the lineages is compatible with the valley’s age. The cover, it is expected that forest dependent organisms first prediction is supported by morphological charac- presented strong demographic expansion in response to ters that diagnose each of the bird populations from the forest advance in the Late Holocene, especially in Minas Gerais and from São Paulo (Albuquerque, regions southwards to the Late Pleistocene northern 1996). The prediction regarding the age of the valley, limit of grassland. Thus, we suggest that the demo- however, is not accepted. According to Petri & Fulfaro graphic expansion of X. f. fuscus lineages is related to (1983), the formation of the valley started in the their geographical expansion that followed the forest Miocene-Pliocene (approximately 15 Mya) and lasted advance in the Holocene, as this is compatible with the until the Early Pleistocene. The estimated divergence estimated expansion dates. The southern lineage pos- time of the two X. f. fuscus lineages was 50 000– sibly expanded from the Serra do Mar mountain range 100 000 years ago, which is too recent to match the or from forest refuges along the Paraná river (Ledru

© 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 91, 73–84 82 G. S. CABANNE ET AL. et al., 1998). On the other hand, X. f. tenuirostris pre- tuto Brasileiro do Meio Ambiente e dos Recursos sented no demographic expansion, possibly because Naturais Renováveis (Brazil), Instituto Florestal de their forest habitat was less affected by Pleistocene cli- São Paulo (Brazil), Instituto Estadual de Florestas de matic alterations (Behling, 1998, 2002). Minas Gerais (Brazil), Ministerio de Ecología de Mis- The hypothesis of refuges, the influence of geogra- iones (Argentina), and G. Patané for the permits to col- phy, and river barriers are among the most discussed lect samples. We are also grateful to J. Patané, R. models in the study of Neotropical diversification. In Pessoa, I. Roesler, and E. Krauczuk for assistance in conclusion, our data did not support the primary influ- field work, E. H. Sari for laboratory assistance, and M. ence of geographical barriers (VPSR) or rivers in the E. Danoviz for the language correction of the manu- divergence between the two main mitochondrial lin- script. D. Meyer and two anonymous reviewers pro- eages of X. f. fuscus of the south-eastern Atlantic vided useful comments. We also thank the Editor J. A. forest. Instead, the data supported the influence of Allen. This study was funded by Fundação de Amparo isolation by geographical distance, recent vicariance à Pesquisa do Estado de São Paulo, Coordenação de events, and demographic expansions in shaping the Aperfeiçoamento de Pessoal de Nível Superior, Con- phylogeographical structure of X. fuscus. These vicar- selho Nacional de Pesquisa, Programa Ecológico de iance and expansions events appear to be related to Longa Duração, and World Wildlife Fund USA. recent natural forest landscape dynamics. 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